Geminiviruses present the most serious disease problem in many vegetable crops in tropical and subtropical regions. For example, major epidemics of geminivirus infections of beans and tomatoes have occurred in Florida, the Caribbean Basin, Mexico, and Central America. In the past, traditional breeding methods failed to produce cultivars with significant levels of resistance to geminiviruses. An alternative approach lies in producing virus-resistant transgenic plants according to the present invention.
The geminivirus group are single stranded DNA viruses that infect both monocotyledonous (monocot) and dicotyledonous (dicot) plants. A common feature among all gemini viruses is the mode of genomic replication, which involves a rolling circle mechanism.
Tomato mottle virus (ToMoV) is one example of a geminivirus. It has a two component (bipartite) genome, an ability to infect dicot plants and is transmitted by whitefly. The DNA of its two genomic components, DNA-A and DNA-B, has previously been cloned and sequenced. Isolated clones of DNA-A and DNA-B of ToMoV are themselves infectious when mechanically inoculated into tomato and N. benthamiana, or when delivered to either host by agroinoculation. An invariant geminiviral DNA sequence required for replication is present in an intergenic, common region (CR) in each genomic component.
The ToMoV DNA-A genomic component has four ORF, one of which, AC1, must be expressed for efficient replication of both A and B components. The AC1 ORF encodes a protein having several functional activities: a DNA binding site specific to the DNA-A CR; a DNA nicking activity; and a NTP binding activity. The DNA binding region mediates an initiator protein function during rolling circle replication.
AC3 protein is a second ToMoV-coded function involved in DNA replication and production of single-stranded circular DNA.
Tomato yellow leaf curl virus (TYLCV) is another example of a geminivirus. TYLCV has a monopartite genome organization, infects monocot plants, and is leaf-hopper transmitted. The TYLCV Cl protein is required for replication, encoded by the C1 ORF.
Being DNA viruses, geminiviruses offer advantages for antiviral strategies. Several geminiviruses have been cloned and sequenced. Transgenic plants having mutant viral genes can be produced, e.g., by introducing expression cassettes comprising mututated virus genes directly into plants with a particle gun, or into plant suspension cells or protoplasts by electroporation, or by Agrobacterium transfection.